PROJECT SUMMARY Microelectrodes are popular for sensing real-time changes in neurotransmitters and understanding the dynamics of neurotransmission in the brain. However, technology has changed little in three decades and there are many unmet technological needs for in vivo electrochemical sensors. In particular, electrodes are needed with high selectivity to discriminate different molecules, small enough tips to localize in small model organisms, and geometries that enable global sensing at high temporal resolution. One new electrode is unlikely to solve all these problems; instead, the electrochemical tool-kit needs to be expanded with many types of electrode designs, materials, and fabrication strategies so that electrodes can be customized for the application. The long term goal of my lab is to develop new electrodes for the measurement of real-time changes of neurotransmitters in vivo and use them to understand real-time detection of neurotransmitter dynamics in the brain. The goal of this project is to develop carbon nanomaterial electrodes, carbon nanopipettes, and 3D printed electrodes with tunable selectivity, tip diameter, and geometry. In the first specific aim, we will use carbon nanomaterials, surface treatments, custom waveforms, and imaging-based software approaches to tune the oxidation of difficult to detect molecules and reduce biofouling. Discrimination and co-detection of histamine, adenosine, and hydrogen peroxide will be targeted, as well as reduced fouling by serotonin and its metabolites. In the second aim, carbon nanopipettes will be developed as nanoelectrodes with tunable tip diameters that can sample from submicron regions, facilitating measurements in small Drosophila brain regions without destroying the tissue. Different geometries will be compared, included closed-tip, cavity, and open tube pipettes. In the third aim, a completely new way to make an electrode will be explored: nano-3D printing. A Nanoscribe 3D printer with 500 nm printing resolution will be used and designs then oxygen/argon annealed, which causes shrinking and carbonization. This 3D printing technique will enable rational design of free-standing, high temporal resolution sensors and flexible carbon mesh electrodes that measure neurotransmitters more globally. The result of this project will be many different kinds of electrodes that enable many different neurochemical applications, from discriminating adenosine and histamine transients in vivo, to dopamine detection in discrete Drosophila regions that are less than 10 ?m wide, to rapid measurements of neurotransmission on a global scale. The significance of this project is that it will transform in vivo microelectrode design to facilitate complex dynamic measurements of neurochemistry that will lead to a better understanding of the how the brain functions and how if malfunctions during disease. The expected positive impact of this new electrode design is thus new platforms of electrodes with tunable electrochemistry to better understand real-time neurotransmission.